Highly cross-linked polymeric supports

ABSTRACT

A polymer useful as a solid support for a variety of applications is provided. The polymer is prepared from a composition comprising at least one type of olefinic monomer and a multifunctional oxyethylene-containing meth(acrylate) crosslinker of the following formula (I): ##STR1## wherein: (a) R 1 , R 2 , and R 3  are each independently hydrogen or a methyl group; 
     (b) R 4  is hydrogen or an organic group or substituent that can interact in the polymerization and/or crosslinking process or is nonreactive under the conditions of the polymerization and/or crosslinking process; and 
     (c) each of l, m, and n is no greater than about 100.

The present invention was made with government support under the National Institutes of Health Grant Nos. GM 42722 and 51628. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

An increasing array of organic chemical reactions, e.g., acylations, alkylations, enolate additions, Wittig reactions, reductions, oxidations, etc., that are traditionally carried out in homogeneous solution are now being adapted for solid-phase synthesis. For this reason, there is a large demand for suitable polymeric supports. For the most well-studied case, i.e., solid-phase peptide synthesis (SPPS), a wide range of materials have been developed.

Merrifield's original studies, and much subsequent work, used low crosslinked polystyrene [Merrifield, J. Am. Chem. Soc., 85, 2149-2154 (1963)]. Polystyrene, however, is limited in the number and variety of solvents that can be used. Because peptide synthesis takes place within a well-solvated gel [Sarin et al., J. Am. Chem. Soc., 102, 5463-5470 (1980)], one aspect that has been considered in the development of supports for SPPS is how well the material swells. To ensure good swelling properties, most polymeric supports used previously for this purpose have had a minimum amount of crosslinking consistent with mechanical stability. Generally, it is believed that the more crosslinking, the more mechanically stable is the polymer, but the less swellable it is. Thus, polymers used as solid supports have been developed to balance swelling with mechanical stability by using a low level of crosslinker (e.g., typically less than about 10 mole-% and usually only about 1-2 mole-%).

For SPPS, another consideration is to produce a support of the same polarity as the peptide backbone. Sheppard and coworkers developed a polyamide material known commercially as PEPSYN [Arshady et al., J. Chem. Soc., Perkin Trans. I, 529-527 (1981)]. This material, however, has not been commercially successful. Another consideration, of particular importance for continuous-flow procedures, is the mechanical stability of the support. Efforts to develop materials for this purpose resulted in PEPSYN K [Atherton et al., J. Chem. Soc., Chem. Commun., 336-337 (1981)] and polyamide-Polyhipe [Small and Sherrington, J. Chem. Sec., Chem. Commun., 1589-1591 (1989)].

Earlier work in the area of solid-phase synthesis also focused on supports that comprise polyethylene glycol (PEG) grafted onto low crosslinked polystyrene (PS). The resultant PEG-PS can be used in continuous-flow processes, has good swelling properties, and shows good compatibility with peptides [Zalipsky et al., React. Polym., 22, 243-258 (1994), and U.S. Pat. No. 5,235,028 (Barany et al.)]. Solid-phase peptide synthesis procedures and supports are summarized, for example, by G. Barany et al., Int. J. Peptide Protein Res,, 30, 705-739 (1987) and J. M. Stewart et al., Innovation and Perspectives in Solid Phase Synthesis: Peptides, Polypeptides, and Oligonucleotides. Macro-organic Reagents and Catalysts, R. Epton, Ed., SPCC UK Ltd., 1-9 (1990).

A need exists for other polymeric supports, particularly for solid-phase synthesis, but also for applications in chromatography, immobilization, etc.

SUMMARY OF THE INVENTION

The polymers of the present invention are prepared from multifunctional oxyethylene-containing (meth)acrylate crosslinkers, and optional secondary crosslinkers, with an olefin-containing monomer, preferably a functionalized olefin-containing monomer. The multifunctional oxyethylene-containing meth(acrylate) crosslinkers used in making the highly crosslinked polymers of the present invention are represented by the following formula (I): ##STR2## wherein R¹, R², and R³ are each independently hydrogen or a methyl group; R⁴ is hydrogen or an organic group or substituent that can interact in the polymerization and/or crosslinking process or is nonreactive under the conditions of the polymerization and/or crosslinking process; and each of l, m, and n is no greater than about 100 (preferably each of l, m, and n is 1-30, and more preferably l+m+n total 5-25). The crosslinkers are polymerized with one or more olefinic monomers optionally functionalized with amino groups, carboxyl groups, hydroxyl groups, etc. The synthesis of the polymers of the present invention is particularly advantageous because it occurs in one step.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HPLC chromatogram evaluating crude ACP(65-74) amide preparation described in Example 10. A VYDAC C₁₈ reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 19:1 to 3:1 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 nm.

FIG. 2. HPLC chromatogram evaluating crude ACP(65-74) amide preparation described in Example 11. A VYDAC C₁₈ reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 19:1 to 3:1 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 nm.

FIG. 3. HPLC chromatogram evaluating crude (Ala)₁₀ -Val amide preparation described in Example 12. A VYDAC C₁₈ reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 1:0 to 6:4 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 nm.

DETAILED DESCRIPTION

The present invention provides highly crosslinked polymers suitable for use in a wide range of applications. For example, the crosslinked polymers of the present invention can be used as solid supports in a variety of solid-phase synthetic applications, as stationary phases in chromatography, and as matrices for immobilization of macromolecules. The polymers of the present invention can be prepared in a wide range of molecular weights. They can also have a wide range of pore sizes, porosities, surface areas, etc., depending on the desired end use. Although they can also be prepared with a wide range of crosslinking, they are preferably highly crosslinked (i.e., prepared using at least about 10 mole-% total crosslinker).

Although the polymers of the present invention are highly crosslinked, they can also possess good swelling properties in various solvents (e.g., dimethylformamide, methylene chloride, acetonitrile, tetrahydrofuran, and water). Preferably, the polymers of the present invention increase in volume by at least about 1.5 times, and more preferably at least about 3 times, in a suitable solvent such as methylene chloride or dimethylformamide. Typically, good swelling properties result from low levels of crosslinking. Thus, the swelling capability of the polymers of the present invention is unexpected. Also, the polymers of the present invention have generally good compatibility with peptides. That is, they have the same polarity, e.g., hydrophobicity/hydrophilicity, as the peptides, which is advantageous in solid-phase peptide synthesis.

The polymers of the present invention are prepared from multifunctional oxyethylene-containing (meth)acrylate crosslinkers, and optional secondary olefin-containing crosslinkers, with an olefin-containing monomer (i.e., oleic monomer), preferably a functionalized olefin-containing monomer, and more preferably an amine-functionalized olefin-containing monomer. The multifunctional oxyethylene-containing meth(acrylate) crosslinkers used in making the highly crosslinked polymers of the present invention are represented by the following formula (I): ##STR3## wherein R¹, R², and R³ are each independently hydrogen or a methyl group; R⁴ is hydrogen or an organic group or substituent that can interact in the polymerization and/or crosslinking process or is nonreactive under the conditions of the polymerization and/or crosslinking process; and each of l, m, and n is no greater than about 100 (preferably each of l, m, and n is 1-30, and more preferably l+m+n total 5-25). It should be understood that each of l, m, and n can be the same or different. This multifunctional acrylate or methacrylate crosslinker containing one or more oxyethylene groups contributes to the unique properties of the polymers of the present invention.

As used herein, an organic group or substituent is nonreactive under the conditions of the polymerization and/or crosslinking process if it does not undergo chemical change or transformation during the reaction and does not prevent the reaction. By this it is meant that the group is selected such that the reactants can react in the manner described. An organic group or substituent interacts in the polymerization and/or crosslinking process if it reacts with the olefinic monomer or other crosslinker molecules to cause chain growth or crosslinking. Suitable R⁴ groups include substituents such as hydroxyl groups, carboxyl groups, amide groups, ester groups, halogens, amine groups, and the like, as well as alkyl groups, aryl groups, alkaryl or aralkyl groups, alkenyl groups, alkynyl groups, and the like, which can optionally include nonperoxidic oxygen, sulfur, or nitrogen atoms, and be unsubstituted or substituted with the substituents listed above. Preferably, R⁴ is hydrogen, an oxyethylene-containing methacrylate group, an alkyl group, or a hydroxyalkyl group. More preferably, R⁴ is hydrogen, --CH₂ --(O--CH₂ --CH₂)_(x) --O--C(O)--C(R⁵)═CH₂ wherein R⁵ is hydrogen or methyl group and x is no greater than about 100 (preferably 1-30), a (C₁ -C₄)alkyl group, a hydroxy(C₁ -C₄)alkyl group. Thus, the multifunctional oxyethylene-containing (meth)acrylate crosslinkers can be tri- or tetra-functional acrylates or methacrylates.

Typically, the polymers of the present invention are prepared using a high level of crosslinker (i.e., at least about 10 mole-%, based on the total number of moles of reactants). Preferably, the polymers of the present invention are prepared using at least about 15 mole-% total crosslinker, more preferably at least about 25 mole-%, and most preferably at least about 50 mole-% total crosslinker. The total amount of crosslinker can be as high as 98 mole-% and still produce a polymer with good swelling properties. The total amount of crosslinker includes the multifunctional oxyethylene-containing (meth)acrylate crosslinkers (I) and any optional secondary olefin-containing crosslinkers.

The secondary olefin-containing crosslinkers include any crosslinkers typically used in crosslinking polymers made from olefinic and/or (meth)acrylate monomers. Generally, such crosslinkers are of the formula H₂ C═CH--R⁶ --HC═CH₂ or H₂ C═C(CH₃)--R⁶ --(H₃ C)C═CH₂, wherein R⁶ is a divalent organic group containing aromatic and/or aliphatic moieties and optional functionalities such as amide groups, carboxyl groups, nonperoxidic oxygen atoms, and the like. Examples of such secondary crosslinkers include, but are not limited to, divinylbenzene, ethylene glycol dimethacrylate [H₂ C═C(CH₃)--C(O)--O--CH₂ --CH₂ --O--C(O)--(CH₃)C═CH₂ ], poly(ethylene glycol-400)-dimethacrylate [H₂ C═C(CH₃)--C(O)--(O--CH₂ --CH₂)₉ --O--C(O)--(CH₃)C═CH₂ ], N,N'-methylenediacrylamide [H₂ C═CH--C(O)--NH--CH₂ NH--C(O)--CH═CH₂ ], N,N'-1,4-phenylenediacrylamide [H₂ C═CH--C(O)--NH--C₆ H₄ --NH--C(O)--CH═CH₂ ], 3,5-bis(acryloylamido)benzoic acid [H₂ C═CH--C(O)--NH--C₆ H₃ (CO₂ H)--NH--C(O)--CH═CH₂ ], N,O-bisacryloyl-L-phenylalaninol [H₂ C═CH--C(O)--NH--CH(CH₂ --C₆ H₅)--CH₂ --O--C(O)--CH═CH₂ ], pentaerythritol triacrylate [formula I wherein l, m, and n each are 0, R¹, R², and R³ are each H, and R⁴ is an OH group], trimethylolpropane trimethacrylate [formula I wherein l, m, and n each are 0, R¹, R², and R³ are each CH₃, and R⁴ is --CH₂ CH₃ group], and pentaerythritol tetraacrylate [formula I wherein l, m, and n each are 0, R¹, R², and R³ are each H, and R⁴ is --CH₂ --O--C(O)--CH═CH₂ ]. Preferably, the secondary olefin-containing crosslinker is selected from the group consisting of a diacrylate, a dimethacrylate, a diacrylamide, a dimethacrylamide, and a divinylbenzene.

The crosslinkers are copolymerized with one or more olefinic monomers optionally functionalized with amino groups, carboxyl groups, hydroxyl groups, etc. Generally, the functional groups serve as starting points for substituents that will be coupled to the polymeric support. These functional groups can be reactive with an organic group that is to be attached to the solid support or it can be modified to be reactive with that group, as through the use of linkers or handles. The functional groups can also impart various desired properties to the polymer, depending on the use of the polymers. For example, if used in ion exchange chromatography, the polymers of the present invention should include charged groups. If used as supports for peptide synthesis, the polymers of the present invention can include amino groups. Preferably, the polymers of the present invention are made using olefinic monomers containing amino functional groups.

Suitable olefins include, for example, vinyl carboxylic acids such as acrylic acid, methacrylic acid, itaconic acid, and 4-vinylbenzoic acid; vinyl esters such as vinyl acetate, vinyl propionate, and vinyl pivalate; allyl esters such as allyl acetate; allyl amines such as allyl amine and allylethylamine; acrylic esters such as methyl acrylate, cyclohexylacrylate, benzylacrylate, isobornyl acrylate, hydroxybutyl acrylate, glycidyl acrylate, and 2-aminoethyl acrylate; methacrylic esters such as methyl methacrylate, butyl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, ethyl methacrylate, glycidyl methacrylate, and 2-aminoethyl methacrylate; vinyl acid halides such as acryloyl chloride and methacryloyl chloride; styreric and substituted styrenes such as 4,ethylstyrene, 4-aminostyrene, dichlorostyrene, chlorostyrene, 4-hydroxystyrene, hydroxymethyl styrene, 4-hydroxy-3-nitro-styrene, 3-hydroxy-4-methoxy-styrene, and vinylbenzyl alcohol; vinyl toluene; heteroaromatic vinyls such as 1-vinylimidazole, 4-vinylpyridine, and 2-vinylpyridine; mono-functional oxyethylene-containing meth(acrylates) such as poly(ethylene glycol) ethyl ether methacrylate [H₂ C═C(CH₃)--C(O)--O--(CH₂ --CH₂ --O)_(q) --CH₂ --CH₃ wherein q=3-5]; hydroxyl-containing (meth)acrylates such as 3-chloro-2-hydroxypropyl (meth)acrylate and hydroxyalkyl (meth)acrylates wherein the akyl moiety contains 2-7 carbon atoms (e.g., 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 2-hydroxybutyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, 5-hydroxypentyl (meth)acrylate, and 2,3-dihydroxypropyl (meth)acrylate); hydroxyl-containing caprolactone (meth)acrylates such as the ring opening addition products of ε-caprolactone with 2-hydroxyethyl (meth)acrylate or 2-hydroxypropyl (meth)acrylate; poly(akylene glycol)(meth)acrylates such as the ring opening addition products of ethylene oxide and/or propylene oxide with (meth)acrylic acid such as diethylene glycol (meth)acrylate, triethylene glycol (meth)acrylate, and polyethylene glycol methacrylate, and polypropylene glycol methacrylate; hydroxyl-containing (meth)acrylamides such as N-(hydroxymethhyl)(meth)acrylamide, N-(1-hydroxyethyl)(meth)acrylamide, N-(2-hydroxyethhyl)(meth)acrylamide, N-methyl-N-(2-hydroxyethyl) (meth)acrylamide, N-(1-hexyl-2-hydroxy-1-methylethyl)(meth)acrylamide, N-propyl-N-(2-hydroxyethyl(meth)acrylamide, N-cyclohexyl-N-(2-hydroxypropyl)(meth)acrylamide, α-bromo-N-(hydroxymethyl)acrylamide, and α-chloro-N-(hydroxymethyl)acrylamide); allyl alcohols such as allyl alcohol, 1-buten-3-ol, 1-penten-3-ol, 1-hexen-3-ol, 1-hydroxy-1-vinyl cyclohexane, 2-bromoallyl alcohol, 2-chloroallyl alcohol, 2-methyl-1-buten-3-ol, 2-ethyl-1-penten-3-ol, and 1-phenyl-2-propen-1-ol; hydroxyl-containing vinyl ethers such as hydroxyethyl vinyl ether and hydroxybutyl vinyl ethers); and hydroxyl-containing allyl ethers such as allyl-1-methyl-2-hydroxyethyl ether, allyl-2-hydroxypropyl ether, allyl-2-hydroxy-l-phenyl ether, and allyl-2-hydroxy-2-phenyl ether. It should be understood that one or more types of olefinic monomers can be used to make the polymers of the present invention. Depending on the end use, one can choose the desired combination of monomers and the desired type and amount of functionalization.

The polymer of the present invention can be made using optional ingredients such as free-radical initiators (e.g., thermolytic and/or photolytic initiators). Free-radical initiators useful in the present invention include those normally suitable for free-radical polymerization of acrylate monomers. These species include azo compounds, tertiary amines, as well as organic peroxides, such as benzoyl peroxide and lauryl peroxide, and other initiators. Examples of azo compounds include 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2-methylpropionitrile), and 2,2'-azobis(isobutyronitrile). Commercial products of this type include VAZO 67, VAZO 64 and VAZO 52 initiators supplied by E.I. duPont de Nemours & Co. Typically about 0.1-2.0 wt-% is used based upon the total monomer weight.

Preparation of the Polymer

The polymer of the present invention, preferably in the form of particles or beads, can be produced by various methods, including bulk polymerization followed by grinding and sieving, or by emulsion polymerization, dispersion polymerization, suspension polymerization, seeded polymerization, or precipitation polymerization. Such techniques are described in R. Arshady, J. Microencapsulation, 5, 101 (1988), R. Arshady et al., J. Chem. Soc., Perkins Trans, I, 529-537 (1981), P. Karda et al., Int. J. Pept. Prot. Res., 38, 385-391 (1991), and P. Reinholdsson et al., Angew, Makromol. Chem., 192, 113-132 (1991).

Whether a bulk process or a suspension polymerization process, the process is advantageous particularly because functionalized crosslinked polymers can be made in essentially one step of reacting a functionalized monomer and a crosslinker containing oxyethylene functionality. Other functionalized crosslinked polymers are known containing oxyethylene functionality, however, they are generally grafted polymers prepared from polyethylene glycol. Generally, the bulk one-step process involves combining one or more types of olefinic monomers with a multifunctional oxyethylene-containing (meth)acrylate crosslinker, optionally with a secondary olefin-containing crosslinker and a free-radical initiator, in a suitable solvent. The solvent is one in which the reactants will substantially dissolve, preferably at ambient temperature (i.e., 25°-30° C.). By substantially dissolving it is meant that the reactants do not have to be completely soluble in the solvent of choice, but they should be sufficiently soluble such that there is enough in solution at the temperature at which the polymerization occurs to effect the polymerization. Suitable solvents include cyclohexanol and higher alcohols, toluene, chloroform, methylene chloride, acetonitrile, tetrahydrofuran, carbon tetrachloride, benzene, and the like. The reaction is typically carded out in a substantially oxygen-free atmosphere (e.g., in the presence of argon or nitrogen). To initiate the polymerization, the reaction mixture can be exposed to a source of energy, such as ultraviolet radiation and/or an elevated temperature, for a time sufficient to effect the polymerization. Preferably, the reaction is carried out at a temperature of about -30°-120° C. (more preferably about 50°-90° C.) and exposed to radiation in the wavelength of about 350-370 nm. The polymer can then be crushed and sieved to the desired particle size.

For beads (e.g., spherical particles), any of three generally known suspension polymerization methods can be used. One of these methods uses conventional suspension agents with optional anionic surfactants, another ("limited coalescence method") uses a negatively charged colloidal silica suspending agent and a water-soluble promoter, and a third ("surfactant method") uses a surfactant as a suspending agent, to produce smaller particle sizes. A variety of solvents can be used, such as water or organic solvents such as cyclohexanol. Generally, the choice of solvent, as well as reaction conditions (e.g., temperature), can affect the properties of the polymer (e.g., porosity, pore size, and surface area).

With respect to the first of the three suspension polymerization methods, suspension stabilizers useful in preparing the particles of the present invention are those conventionally used in suspension polymerization processes. The terms "suspension stabilizers," "suspending agents," and "suspension agents" are used interchangeably herein. They may be minimally water-soluble inorganic salts such as those selected from the group consisting of tribasic calcium phosphate, calcium carbonate, calcium sulfate, barium sulfate, barium phosphate, magnesium carbonate, and mixtures thereof. Preferred inorganic suspending agents include those selected from the group consisting of barium sulfate, tribasic calcium phosphate, and mixtures thereof. Water-soluble organic suspending agents, such as those selected from the group consisting of polyvinyl alcohol, poly-N-vinylpyrrolidone, polyacrylic acid, polyacrylamide, and hydroxyalkyl cellulose, can also be used. Surfactants can also be used in this method. Typically, anionic surfactants such as sodium lauryl sulfate and sodium dioctyl sulfosuccinate, are used.

When polymeric particles of less than one micron in diameter are desired, a surfactant or emulsifying agent alone is used as the suspending agent. Surfactants or emulsifiers useful in this "surfactant method" are typically anionic surfactants, cationic surfactants, or nonionic surfactants. Anionic surfactants useful in the present invention include, but are not limited to, the group consisting of alcohol sulfates, alkylaryl sulfonates, ethoxylated alkyl phenol sulfates, ethoxylated alkyl phenol sulfonates and mixtures thereof. Cationic surfactants include, but are not limited to, the group consisting of quaternary ammonium salts wherein at least one higher molecular weight group and two or three lower molecular weight groups are linked to a common nitrogen atom to produce a cation, and wherein the electrically balancing anion is selected from the group consisting of a halide (bromide, chloride, etc.), acetate, nitrite, and lower alkyosulfonate (methosulfate, ethyosulfate, etc.), and mixtures thereof. Nonionic surfactants useful in the present invention include, but are not limited to, the group consisting of ethoxylated alkyl phenols, ethoxylated fatty acids, and ethoxylated fatty alcohols and mixtures thereof. A combination of more than one surfactant or emulsifier is also found to be useful in the invention.

The polymeric particles of the present invention can also be produced by the limited coalescence method. Typically, a reaction mixture of monomers, a negatively charged colloidal silica suspending agent, and a free-radical initiator, are stirred in water under high-speed agitation conditions to break the monomer phase into small droplets. The stirred suspension is heated under nitrogen while polymerization takes place and the desired particles are formed. The particles are collected and washed with water, then dried. The colloidal silica particles have dimensions of about 1-100 nanometers and preferably of about 5-70 nanometers. The size and concentration of these particles controls the size of the polymer particles. Smaller silica particles and higher silica concentration provides smaller bead diameters. Hydrophilic colloidal silica useful as the suspending agent is available commercially, for example, under the tradenames and in the particle sizes as follows: LUDOX TM (20 nm); LUDOX HS (12 nm); LUDOX SM (7 nm); and LUDOX AM (12 nm); all supplied by E.I. duPont de Nemours Company; and NALCO 1060 (60 nm) supplied by Nalco Chemical Company, Oakbrook, Ill.

In this method, the negatively charged colloidal silica suspending agent is preferably used with a water-soluble "promoter" that affects the hydrophobic-hydrophilic balance of the colloidal particles. The components of the promoter are chosen to ensure good water solubility and sufficient complexing with colloidal silica. More specifically, the promoter forms a complex with the suspending agent which is less hydrophilic than the colloidal particles themselves. Although the inventors do not wish to be held to any particular theory, it is believed that the promoter drives the particles of the colloid to the liquid-liquid interface of the oleophilic or hydrophobic droplets and the aqueous medium. As a consequence, the complex is compatible with the hydrophobic or oleophilic monomers dispersed in the aqueous reaction medium. The complex coats the monomer droplets and inhibits their coalescence. Typically, about 0.02-0.5 percent by weight of a promoter is used based on the weight of the aqueous phase. The water-soluble promoter is preferably a low-molecular weight (i.e., about 200 to about 1000 number average molecular weight) organic condensation polymer of a lower alkylene dicarboxylic acid and an alkanol amine (preferably a mono- or dialkanol amine). The dicarboxylic acid can have an alkylene chain having about 2-6 carbon atoms in length. The preferred diacid of this class is adipic acid. The alkanol amine preferably is a lower alkanol amine of which the alkanol groups contain about 1-4 carbon atoms, selected from the group consisting of diethanolamine, 2-amino-2-ethyl-1,3-propanediol, methyl amino ethanol, N-methyldiethanolamine, N-propyldiethanolamine and N-butyldiethanolamine. With adipic acid, these alkanol amines form the polyesters (by which term we also include polyesteramides), such as poly(diethanolamine adipate) and poly(methylamino ethanol adipate). Preferably, the water-soluble promoter is a condensation polymer of adipic acid and diethanolamine.

Also desirable in the polymerization reaction mixture is an inhibitor, i.e., a water-soluble substance to prevent the emulsion or solution polymerization of the monomers in the aqueous phase. Examples of inhibitors include, but are not limited to, those selected from the group consisting of sodium nitrite, copper salts, methylene blue, potassium dichromate, phenols, and mixtures thereof. A preferred example of such a water-soluble polymerization inhibitor is potassium dichromate. Typically about 0.01-0.1 percent by weight of an inhibitor is used based on the total weight of the aqueous phase.

Applications

The polymer of the present invention can be used as a support for a variety of applications. These include, for example, solid-phase synthesis, chromatography (e.g., affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, ion-pair chromatography, normal-phase chromatography, reversed-phase chromatography, and hydrophobic interaction chromatography), and immobilization of macromolecules (e.g., enzymes and antibodies) to increase their stability. The polymeric supports of the present invention have several desirable characteristics for these applications, particularly for solid-phase synthesis: they swell in a variety of solvents; they are stable under the conditions used in most solid-phase syntheses; and they are stable at the pressures experienced in column reactors.

Solid-phase peptide synthesis typically begins with covalent attachment of a first amino acid (e.g., an "internal reference" amino acid) to the solid support. If the solid support was prepared with amino-functional monomers, the carboxyl group of an N.sup.α -protected amino acid is covalently linked to a handle moiety which is attached to the amino group on the polymer. A "handle" is defined as a bifunctional spacer which serves to attach the initial amino acid residue to the polymeric support. One end of the handle incorporates a smoothly cleavable protecting group and the other end of the handle couples to the functionalized solid support. Handles which can be used with the present polymers in solid-phase peptide synthesis include, for example acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al., J. Org. Chem., 55, 3730-3743 (1990) and references cited therein, and in U.S. Patent Nos. 5,117,009 (Barany) and 5,196,566 (Barany et al.). The appropriate handles are coupled quantitatively in a single step onto the amino-functionalized supports to provide a general starting point of well-defined structures for peptide chain assembly. The handle protecting group is removed and the C-terminal residue of the N.sup.α -protected first amino acid is coupled quantitatively to the handle. Once the handle is coupled to the solid-phase and the initial amino acid or peptide is attached to the handle, the general synthesis cycle proceeds. The synthesis cycle generally consists of deprotection of the N.sup.α -amino group of the amino acid or peptide on the resin, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next N.sup.α -protected amino acid. The cycle is repeated to form the peptide or protein of interest. Solid-phase peptide synthesis methods using functionalized insoluble supports are well known. Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Barany and Merrifield, In Peptides, Vol. 2, pp. 1-284 (1979); Barany et al., Int. J. Peptide Protein Res,, 30, 705-739 (1987). It should be understood that the polymeric materials of the present invention can be used in the solid-phase synthesis of a variety of organic molecules. Peptides are used herein merely as exemplary such molecules.

In one particular embodiment of the present invention, an internal reference amino acid (Fmoc-Nle-OH) was coupled to the free amino groups on the resin particles, followed by deprotection and coupling of the handle Fmoc-5-(-4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL). The challenging sequence 65-74 of acyl carrier protein (ACP) was synthesized successfully on the support by Fmoc-chemistry, using both a conventional solvent (DMF, FIG. 1) and a non-conventional solvent (acetonitrile, FIG. 2) as the reaction media. In addition, H-(Ala)₁₀ -Val amide (FIG. 3) has been synthesized with results comparable to those obtained on commercially available PEG-PS.

Objects and advantages of this invention are further illustrated by the following examples. The particular materials and amounts thereof recited in these examples as well as other conditions and details, should not be construed to unduly limit this invention. All materials are commercially available from Aldrich Chemical Company, Inc., Milwaukee, Wis., except where stated or otherwise made apparent.

EXAMPLES Example 1-7: Preparation of Polymers Example 1

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/poly(ethylene glycol-400)-dimethacrylate/2-aminoethyl methacrylate-copolymer: 2-aminoethyl methacrylate.HCl (0.60 g, 3.6 mmol, obtained from Eastman Kodak, Rochester, N.Y.), poly(ethylene glycol-400)-dimethacrylate (3.88 g, 7.0 mmol), trimethylolpropane ethoxylate ("14/3 EO/OH") triacrylate (structure I, l+m+n˜14 (divided among 3 acrylate chains), R¹, R², R³ =H, R⁴ =C₂ H₅) (2.74 g, 3.0 mmol) and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 16 ml of cyclohexanol. The solution, in a KIMAX Screw Cap Culture Tube, was sonicated and then purged with a stream of nitrogen for 5 minutes. The tube was sealed and placed in a RAYONET Photochemical Reactor overnight. The polymerization was initiated by a combination of photolytic (350 nm) and thermolytic (50° C.) dissociation of 2,2'-azobis(2-methylpropionitrile). The resulting polymer was ground in a mortar and wet-sieved with water through 106 μm and 125 μm sieves. The 106-125 μm fraction was collected and washed with water (acidified with trifluoroacetic acid "TFA" to pH 1-2), and methanol on a sintered glass funnel. The resin (2.66 g) was dried in vacuo overnight. A portion of the resin was derivatized as described in Examples 8 and 9 with an internal reference and then a handle. The loading of Fmoc-PAL on the resulting resin was 0.25 mmol per gram of resin.

Example 2

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/poly(ethylene glycol-400)-dimethacrylate/allylamine copolymer: allylamine (0.16 g, 2.75 mmol), poly(ethylene glycol-400)-dimethacrylate (3.88 g, 7.0 mmol), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (2.74 g, 3.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 12 ml of cyclohexanol. The solution was sonicated, purged with a stream of nitrogen for 5 minutes and then polymerized and processed as described in Example 1 to give 1.88 grams of resin. A portion of the resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.11 mmol per gram of resin.

Example 3

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/4-aminostyrene copolymer: 4-aminostyrene (0.41 g, 3.45 mmol, obtained from Lancaster, Windham, N.H.), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (8.21 g, 9.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 12 ml of cyclohexanol. The solution was sonicated, purged with a stream of nitrogen for 5 minutes, and then polymerized and processed as described in Example 1 to give 2.35 grams of resin. A portion of the resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.22 mmol per gram of resin.

Example 4

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH)/allylamine copolymer: allylamine (0.514 g, 9.0 mmol), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (8.208 g, 9.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 17.5 ml of cyclohexanol. The solution was sonicated, purged with a stream of nitrogen for 5 minutes and then polymerized and processed as described in Example 1 to give 2.26 g resin. A portion of the resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.29 mmol per gram of resin.

Example 5

Preparation of parent and amino-functionalized trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/acrylic acid copolymer: acrylic acid (0.25 g, 3.45 mmol), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (8.21 g, 9.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 12 ml of cyclohexanol. The solution was sonicated, purged with a stream of nitrogen for 5 minutes, and then polymerized and processed as described in Example 1 to give 2.40 grams of resin.

A portion (0.40 g) of this ground and sieved trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/acrylic acid copolymer was treated with N,N'-diisopropylcarbodiimide (40 mg, 0.32 mmol, "DIPCDI" obtained from Advanced ChemTech, Louisville, Ky.) and ethylenediamine (99 mg, 1.64 mmol) in 6 ml of DMF-CH₂ Cl₂ (1:1, v/v) and allowed to react at 25° C. overnight. The polymer particles were washed with dimethylformamide "DMF", methanol, acetonitrile and methylene chloride, and then dried in vacuo overnight. The resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.15 mmol per gram of resin.

Example 6

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/poly(ethylene glycol) ethyl ether methacrylate/2-aminoethyl methacrylate-copolymer: 2-aminoethyl methacrylate.HCl (0.73 g, 3.6 mmol), poly(ethylene glycol) ethyl ether methacrylate (0.74 g, 16 mmol), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (7.30 g, 8.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 20 ml of cyclohexanol. The solution was sonicated, purged with a stream of nitrogen for 5 minutes, and then polymerized and processed as described in Example 1 to give 1.84 gram of resin. The resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.27 mmol per gram of resin.

Example 7

Preparation of trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate/methyl methacrylate/2-aminoethyl methacrylate-copolymer: 2-aminoethyl methacrylate.HCl (0.56 g, 3.4 mmol), methyl methacrylate (0.47 g, 4.6 mmol), trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate (7.30 g, 8.0 mmol), and 2,2'-azobis(2-methylpropionitrile) (0.1 g, 0.6 mmol) were dissolved in 14 ml of cyclohexanol-water (13:1, v/v). The solution was sonicated, purged with a stream of nitrogen for 5 minutes, and then polymerized and processed as described in Example 1. A portion of the resin was derivatized as described in Examples 8 and 9. The loading of Fmoc-PAL on the resulting resin was 0.20 mmol per gram.

Example 8-9: Derivatization of the Polymers (Coupling of "Internal Reference" Amino Acid and Handle) for Solid-Phase Synthesis Example 8

Coupling of Fmoc-Nle-OH (Fmoc-norleucine; internal reference amino acid) to amino-functionalized resin: Fmoc-Nle-OH (255 mg, 0.72 mmol, obtained from Chem-Index International, Wood Dale, Ill.) in DMF (2 ml), N,N'-diisopropylcarbodiimide (91 mg, 0.72 mmol) in DMF (0.5 ml), and 1-hydroxy-7-azabenzotriazole (98 mg, 0.72 mmol) in DMF (0.5 ml) were added to 0.6 g of resin and reacted at 25° C. overnight. The resin was washed with DMF (5×6 ml) and methylene chloride (5×6 ml). The Fmoc-group was removed from the Fmoc-Nle-resin by treatment with piperidine-DMF (1:4, v/v) for 15+25 min, and then again washed with DMF (5×6 ml) and methylene chloride (5×6 ml).

Example 9

Coupling of Fmoc-PAL-OH [Fmoc-5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid] to Nle-functionalized resin: Fmoc-PAL-OH (354 mg, 0.72 mmol, Millipore, Bedford, Mass.) in DMF (2 ml), N,N'-diisopropylcarbodiimide (91 mg, 0.72 mmol) in DMF (0.5 ml) and 1-hydroxy-7-azabenzotriazole (HOAt) (98 mg, 0.72 mmol) in DMF (0.5 ml) were added to 0.6 g of Nle-resin (product of Example 8) and reacted at 25° C. overnight. The resin was washed with DMF (5×6 ml) and methylene chloride (5×6 ml).

Example 10-12: Peptide Synthesis on the Supports Example 10

Synthesis of acyl carrier protein (ACP) (65-74) amide (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂) on PAL-Nle-functionalized resin: the synthesis was performed intentionally under non-optimized conditions, in order to emphasize the usefulness of this new class of supports. For this Example 10, Fmoc-PAL-Nle-resin (0.1 g) prepared according to Example 4, was used. Fmoc-removal was achieved with piperidine-DMF (1:4, v/v) for 5+15 minutes (i.e., this was carried out in two steps, one for 5 minutes and one for 15 minutes with the use of fresh piperidine-DMF in the second step). Appropriate N.sup.α -Fmoc-amino acids (150 μmol) were each in mm dissolved in DMF (0.4 ml), and 2 hour couplings were each initiated with 150 μmol of 1-hydroxy-7-azabenzotriazole ("HOAt" obtained from Millipore, Bedford, Mass.) dissolved in DMF (0.2 ml) and 150 μmol of N,Nμ-diisopropylcarbodiimide dissolved in DMF (0.2 ml). All washings between couplings and deprotections were performed with DMF. Cleavage of the peptide was achieved with TFA-CH₂ Cl₂ (9:1, v/v) (2 ml) for 2.5 hours. The solutions containing cleaved peptides were expressed from the solid-phase reaction vessels with positive nitrogen pressure, and the cleaved resins were washed further with TFA-CH₂ Cl₂ (9:1, v/v) (3×2 ml). The combined filtrates were evaporated to dryness (including chasing with CH₂ Cl₂, 3×3 ml), suspended in water, and lyophilized. The crude peptide was dissolved in 0.1% TFA in water-acetonitrile (1:1), and shown to be 91% pure by high-performance liquid chromatography (FIG. 1). A VYDAC C₁₈ reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 19:1 to 3:1 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 nm.

Example 11

Synthesis of acyl carrier protein (ACP) (65-74) amide (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂) on PAL-Nle-functionalized resin: the synthesis was performed intentionally under non-optimized conditions in a, for peptide synthesis, non-conventional reaction medium (acetonitrile), in order to emphasize the usefulness of this new class of supports. For this Example 11, Fmoc-PAL-Nle-resin (0.1 g) prepared according to Example 4, was used. Fmoc-removal was achieved with piperidine-DMF (1:4, v/v) for 5+15 minutes. Appropriate N.sup.α -Fmoc-amino acids (150 μmol) were each in mm dissolved in acetonitrile (0.4 ml, a few drops of DMF was added if needed to dissolve the amino acid derivative), and 2 hour couplings were each initiated with 150 μmol of N,N'-diisopropylcarbodiimide dissolved in acetonitrile (0.4 ml). All washings between couplings and deprotections were performed with acetonitrile. Cleavage of the peptide was achieved with TFA-CH₂ Cl₂ (9:1, v/v) (2 ml) for 2.5 hours. The solutions containing cleaved peptides were expressed from the solid-phase reaction vessels with positive nitrogen pressure, and the cleaved resins were washed further with TFA-CH₂ Cl₂ (9:1, v/v) (3×2 ml). The combined filtrates were evaporated to dryness (including chasing with CH₂ Cl₂, 3×3 ml), suspended in water, and lyophilized. The crude peptide was dissolved in 0.1% TFA in water-acetonitrile (1:1), and shown to be 95% pure by high-performance liquid chromatography (FIG. 2). A VYDAC Gig reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 19:1 to 3:1 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 mm.

Example 12

Synthesis of deca-alanyl-valine amide (H-(Ala)₁₀ -Val-NH₂) on PAL-Nle-functionalized polymer particles: the synthesis was performed intentionally under non-optimized conditions, in order to emphasize the usefulness of this new class of supports. For this Example 12, Fmoc-PAL-Nle-resin prepared according to Example 1, was used. Fmoc-removal was achieved with piperidine-DMF (1:4, v/v) for 3+17 minutes. Appropriate N.sup.α -Fmoc-amino acids (Fmoc-Val-OH and Fmoc-Ala-OH, 120 μmol) in DMF (0.5 riff) were added to 0.1 g resin from Example 1, and 50 minute couplings were initiated with 114 μmol of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ("HATU" obtained from Millipore, Bedford, Mass.) in DMF (0.5 ml), 120 μmol of 1-hydroxy-7-azabenzotriazole ("HOAt") in DMF (0.5 ml) and 230 μmol of N,N-diisopropylethylamine ("DIEA") in DMF (0.5 ml). Cleavage of the peptide was achieved with TFA-water (9:1, v/v) (2 ml) for 2 hours. The solutions containing cleaved peptides were expressed from the solid-phase reaction vessels with positive nitrogen pressure, and the cleaved resins were washed further with TFA-water (9:1, v/v) (3×2 ml). The combined filtrates were evaporated (including chasing with CH₂ Cl₂, 3×3 ml), suspended in water, and lyophilized. The crude peptide was dissolved in neat TFA and shown to be 57% pure by high-performance liquid chromatography (FIG. 3). A VYDAC C₁₈ reversed-phase column (4.6×250 mm) was eluted at a flow-rate of 1 ml/minute with a linear gradient over 30 minutes from 1:0 to 6:4 of 0.1% TFA in water and 0.1% TFA in acetonitrile. Peptides were detected spectrophotometrically at 220 nm.

The results of Examples 10-12 demonstrate that these supports can be used for the synthesis of peptides that are difficult to make in solid-phase.

The complete disclosures of all patents, patent documents, and publications are incorporated herein by reference, as if individually incorporated. While this invention has been described in connection with specific embodiments, it should be understood that it is capable of further modification. The claims herein are intended to cover those variations which one skilled in the art would recognize as the chemical equivalent of what has been described herein. Thus, various omissions, modifications, and changes to the principles described herein may be made by one skilled in the art without departing from the true scope and spirit of the invention which is indicated by the following claims. 

What is claimed:
 1. A polymer prepared from a composition comprising at least one olefinic monomer and a multifunctional oxyethylene-containing meth(acrylate) crosslinker of the following formula (I): ##STR4## wherein: (a) R¹, R², and R³ are each independently hydrogen or a methyl group;(b) R⁴ is hydrogen or an organic group or substituent that can interact in the polymerization and/or crosslinking process or is nonreactive under the conditions of the polymerization and/or crosslinking process; (c) each of l, m, and n is no greater than about 100 with the proviso that at least one of l, m, or n is at least 1; and (d) wherein the total amount of crosslinker used is a least about 50 mole-%.
 2. The polymer of claim 1 wherein the olefinic monomer is functionalized.
 3. The polymer of claim 2 wherein the olefinic monomer is functionalized with an amino group.
 4. The polymer of claim 3 wherein the amino functionalized olefinic monomer is allyl amine, 2-aminoethyl methacrylate, or 4-aminostyrene.
 5. The polymer of claim 1 wherein each of l, m, and n is 1-30.
 6. The polymer of claim 1 wherein the multifunctional oxyethylene-containing meth(acrylate) crosslinker is used in an mount of at least about 10 mole-%.
 7. The polymer of claim 1 wherein the multifunctional oxyethylene-containing meth(acrylate) crosslinker is used in an amount of at least about 25 mole-%.
 8. The polymer of claim 1 wherein the composition further comprises a secondary olefin-containing crosslinker.
 9. The polymer of claim 8 wherein the secondary crosslinker is selected from the group consisting of a diacrylate, a dimethacrylate, a diacrylamide, a dimethacrylamide, and a divinylbenzene.
 10. The polymer of claim 1 wherein each of l, m, and n is 1-100.
 11. The polymer of claim 1 which is in the form of beads.
 12. The polymer of claim 1 which increases in volume by at least about 1.5 times in methylene chloride.
 13. The polymer of claim 12 which increases in volume by at least about 3 times in methylene chloride.
 14. The polymer of claim 1 which is suitable for use in solid phase organic synthesis.
 15. Polymer beads prepared from a composition comprising at least one olefinic monomer and a multifunctional oxyethylene-containing meth(acrylate) crosslinker of the following formula (I): ##STR5## wherein: (a) R¹, R², and R³ are each independently hydrogen or a methyl group;(b) R⁴ is hydrogen or an organic group or substituent that can interact in the polymerization and/or crosslinking process or is nonreactive under the conditions of the polymerization and/or crosslinking process; (c) each of l, m, and n is 1-100; and (d) wherein the total mount of crosslinker used is a least about 50 mole-%.
 16. The polymer beads of claim 15 wherein the olefinic monomer is functionalized.
 17. The polymer beads of claim 16 wherein the olefinic monomer is functionalized with an amino group.
 18. The polymer of claim 17 wherein the amino functionalized olefinic monomer is allyl amine, 2-aminoethyl methacrylate, or 4-aminostyrene.
 19. The polymer of claim 15 wherein each of l, m, and n is 1-30.
 20. The polymer of claim 15 wherein the multifunctional oxyethylene-containing meth(acrylate) crosslinker of formula (I) is used in an mount of at least about 10 mole-%.
 21. The polymer of claim 15 wherein the multifunctional oxyethylene-containing meth(acrylate) crosslinker of formula (I) is used in an amount of at least about 25 mole-%.
 22. The polymer of claim 15 wherein the composition further comprises a secondary olefin-containing crosslinker.
 23. The polymer of claim 22 wherein the secondary crosslinker is selected from the group consisting of a diacrylate, a dimethacrylate, a diacrylamide, a dimethacrylamide, and a divinylbenzene.
 24. The polymer beads of claim 15 which increase in volume by at least about 1.5 times.
 25. The polymer beads of claim 24 which increase in volume by at least about 3 times.
 26. The polymer beads of claim 15 which are suitable for use in solid phase organic synthesis. 